The present invention relates to a composition-of-matter which includes a double stranded RNA (dsRNA) molecule associated with a targeting moiety selected capable of targeting to a specific cell and/or tissue type and to uses of such compositions-of-matter for killing a specific target cell and/or tissue type. Diseases associated with cells/tissue displaying a specific surface marker, such as central nervous system malignancies, include numerous diseases having vast medical and economic impact for which no satisfactory treatment is available.
Malignant gliomas, the most common adult-onset neurological neoplasms, encompass a family of primary central nervous system tumors including glioblastoma, astrocytoma, oligodendroglioma, and ependymoma, along with the juvenile onset neoplasms such as juvenile pilocystic astrocytoma.
Malignant gliomas are typically characterized by overexpression of growth factors/tumor associated antigens believed to significantly contribute to the unchecked growth of such tumors. Various malignant gliomas, such as glioblastomas, exhibit epidermal growth factor receptor (EGFR) overexpression leading to increased aggressiveness and poor prognosis (Kleihues P. and Ohgaki H., 1999. Neuro-oncol. 1999 January; 1(1):44-51; Krishnan et al., 2003. Front Biosci. 8:el-13). Malignant gliomas may also display overexpression of platelet-derived growth factor receptor (Shapiro W R. and Shapiro J R., 1998. Oncology (Huntingt). February; 12(2):233-40; Feldkamp et al., 1997. J Neurooncol. 1997 December; 35(3):223-48), a phenomenon which has also been correlated with increased malignancy and poor prognosis.
Malignant gliomas, the most common type of primary brain tumors, are aggressive, highly invasive, and neurologically destructive tumors which are among the deadliest of all human cancers. Of the estimated 17,000 new brain tumors diagnosed each year in the United States, about half are malignant gliomas. Malignant glioma cells produce very invasive brain tumors with infiltration of both white and gray matter (Bjerkvig et al., 1986. Cancer Res. 46:4071-912). At the time of diagnosis, microscopic extension through much of the neural axis by malignant glioma is the rule (Burger et al., 1980. Cancer 46:1179-86; Kelly et al., 1987. J. Neurosurg. 66:865-74; Moser, 1988. Cancer 62:381-90; and Salazar et al., 1976. Int. J. Radiat. Oncol. Biol. Phys. 1:627-37). Such extension by motile invading cells underlies the incurability by surgery of most gliomas, even when they appear small and restricted in nature. Glioblastoma multiforme (GBM), the most serious form of malignant glioma, are extremely aggressive brain tumors which generally arise in the upper brain (cerebrum), but which may also occur elsewhere in the central nervous system, such as in the spinal cord, cerebellum, brain stem, or optic chiasm. Low-grade gliomas, which include astrocytomas, oligodendrogliomas, and pilocytic astrocytomas, account for 25% of all primary brain tumors, and over time most of these low-grade tumors dedifferentiate into more malignant gliomas. Diffuse astrocytomas are predominantly located in the cerebral hemispheres of adults and have an inherent tendency to progress to anaplastic astrocytoma and (secondary) glioblastoma. The majority of glioblastomas develop de novo (primary glioblastomas), without an identifiable less-malignant precursor lesion (Kleihues P. and Ohgaki H., 2000. Toxicol Pathol. 2000 January-February; 28(1):164-70). No significant therapeutic advances have been made in treatment of malignant gliomas since the landmark Brain Tumor Cooperative Group studies over 20 years ago demonstrated a survival advantage for patients with malignant gliomas who received radiation and single agent chemotherapy (Walker et al., 1978. J. Neurosurg. 49:333-43; and Walker et al., 1980. NEJM 303:1323-9). While cutting edge molecular technologies have led to a better understanding of glioma biology, these have not yet yielded clinical dividends. Glioblastoma in particular is characterized by resistance to standard treatment modalities including surgery, radiation therapy and chemotherapy. While radiation therapy is the standard treatment after surgical resection, these tumors invariably recur and are associated with a uniformly dismal prognosis, and despite several decades of technological advances in neuro-surgery and radiation therapy there has been no significant change to the overall statistics, with the median survival of patients diagnosed with glioblastoma ranging from 9 to 12 months.
Due to the highly restricted localization of malignant gliomas to the central nervous system and the fact that these tumors do not generally generate remote metastases, various gene therapy approaches have been suggested for their treatment (Bansal, K., and Engelhard, H. H., 2000. Curr Oncol Rep 2, 463-72; Shir, A., and Levitzki, A., 2001. Cell Mol Neurobiol 21, 645-56). While prior art gene therapy approaches involving co-injection of gene expression vectors simultaneously with tumor cells may display a measure of effectiveness when tested in-vitro and in-vivo, such approaches have failed to demonstrate satisfactory effectiveness when tested against established tumors, such as would be the case in the clinical setting. Prior art gene therapy approaches employing viral vectors for treatment of glioblastoma have failed to demonstrate satisfactory infection efficiency. This is thought to be due to the histological structure of glioblastoma which is a highly dense tumor, almost completely impermeable to penetration by particles the size of viruses or larger.
Hence, there is a long-felt and urgent need for novel and optimal methods of selectively killing disease associated cells displaying a specific surface marker, such as glioblastoma cells.
One of the mechanisms which virally infected cells employ to protect the body from infection involves triggering of apoptosis by dsRNA molecules which are generally and exclusively expressed in virally infected cells. Virally induced generation of dsRNA leads to up-regulation of interferon (IFN)-α/-β expression. Interferon-α/β are strong anti proliferative cytokines whose mechanism of action involves inducing expression of PKR and the 2′-5′ OAS system for preventing the spread of the virus to cells adjacent to the infected cells. The enzyme PKR is a Ser/Thr protein kinase which upon activation by dsRNA phosphorylates the a subunit of protein synthesis initiation factor eIF-2. This results in sequestration of GDP/GTP exchange factor eIF-2B and rapid inhibition of translation initiation (Farrell et al., 1978. Proc Natl Acad Sci USA 75, 5893-7). Activation of PKR strongly induces cell death by apoptosis which is partly driven by inhibition of the protein synthetic machinery (Jagus, R. et al., 1999. Int J Biochem Cell Biol. 31, 123-38). It is likely that activation of NF-κB, also induced by PKR, prevents immediate cell death allowing production of IFN-α/β. Mechanisms involved in the pro-apoptotic activity of dsRNA also include activation by dsRNA of the 2′-5′ Oligo A synthetase/RNase L system which contributes to the shut off of protein synthesis (Player, M. R., and Torrence, P. F., 1998. Pharmacol Ther 78, 55-113), activation of the stress kinases JNK and p38 (Iordanov, M. S. et al., 2000. Mol Cell Biol. 20, 617-27), activation of transcription factors IRF3 and DRAF1 leading to enhanced expression of several proapoptotic genes, and activation of expression of NO synthetase leading to production of NO and subsequent cell death. Hence, dsRNA is a highly potent anti-proliferative/cytotoxic molecule capable of killing cells via multiple mechanisms (FIG. 1).
Thus, an optimal strategy for treating diseases, such as malignant glioma, would be to use dsRNA to kill disease associated cells/tissues.
Several prior art approaches have been employed or suggested in order to use dsRNA for selectively killing disease associated cells or tissues, such as malignant glioma cells or tissues.
One approach involves administering polylysine/carboxymethylcellulose stabilized pIC intramuscularly to malignant glioma patients (Salazar, A. M. et al., 1996. Neurosurgery 38, 1096-103).
Another approach involves administering polylysine/carboxymethylcellulose stabilized pIC intravenously to glioblastoma, astrocytoma and ependymoma patients (Nakamura, O. et al., 1982. No To Shinkei 34, 267-73).
Yet another approach involves treating nude mice bearing established intracranial tumors derived from a human glioblastoma cell line by tumor-proximal injection of plasmid vector encoding an anti-sense RNA complementary to RNA transcript of Δ(2-7) EGFR, a mutant form of EGFR specifically expressed in glioblastoma, so as to generate an intracellular dsRNA capable of specifically inducing apoptosis of such cells (Ogris, M. et al., 2001. AAPS PharmSci 3:E21).
All of the aforementioned approaches, however, suffer from significant disadvantages. Namely, approaches employing administration of dsRNA to human patients failed to demonstrate optimal therapeutic results, involve the risk of toxic side-effects such as fever, hypotension and leucopenia, and approaches employing mouse models of human disease failed to demonstrate optimal therapeutic effect, have not demonstrated effectiveness in human patients, and are limited to killing of disease associated cells expressing a highly specific mutant RNA. Moreover, none of these approaches are of optimal safety since none of these have the capacity to selectively target the therapeutic reagent exclusively to the target cells, thereby risking unpredictable and potentially harmful side-effects by interaction with non-targeted cells/tissues.
Thus, all prior art approaches have failed to provide an adequate solution for using dsRNA for killing disease associated cells or tissues.
There is thus a widely recognized need for, and it would be highly advantageous to have, a method of using dsRNA for killing disease associated cells or tissues devoid of the above limitation.